Acylated gelatins and their preparations



United States Patent '6 ACYLATEDGELATINS AND THEIR PREPARATIONS Dee Tourteilotte, 'Haddonfield, N. 3., and Henry E. "Williarns, Hollis, N. H., assignors to Charles B. Knox Gelatine Co, Inc., a corporation of New York No Drawing. Application September 24,1954 Serial N0.'-458,286

25 Claims. (Cl. 167-78) The present invention relates to the preparation and purification of derivatives of gelatin'having physicalfand chemical properties that make them useful as blood plasma substitutes for bloodplasma volume extenders 'as well as for many other purposes.

This application is a continuation-impart of our copending applications, "Serial Nos. 268,128 and 280,372, filed January '24, 1952, and April 3, 1952, respectively. Both"of'saidapplicati'ons are now abandoned.

it has been well established that properly prepared gelatin solutions made sterile, pyrogen and antigen free, and with certain considerations'for maintaining'a relatively high average molecular size with controlled colloid osmotic pressure, have been useful for parenteral use-incontrolling circulating blood volume in the experimental and clinical management of shock either from hemorrhages and burns or traumatic and psychogenic temperature (70F). The reduction of average molecular size of the gelatin that is brought about in such a process is generally accompanied by a 'veryisignificant reduction in the retention 'period of 'such materials when they are injected into the intact blood vascular system.

2) The coupling of gelatin molecules into larger complexes by means of chemical treatmentwith the dialde- 'hyde, glyoxal, followed by oxidation with H O ;for.increasing the number'of ionizable groups at pH 7.3 has been attempted withsome success. The reaction hasbeen difiicult to control and results in 'a finished preparation probably having greater'moleculer heterogenicity" than that of the untreated gelatin.

' Gelatin isa derived protein composed of various amino acids linked between adjacent imino and carbonyl groups to provide the classical peptide bond. 'The molecular weight of fgelatinis characteristically large (values of the average molecular weight'vary fromap'proximatly 10,000 to 160,000) and is significantly heterogeneous within any-"particular -'type and grade. "Gelatin" is :com-

posed ofasymmetn'c or rodlike'types ofmolecules, resulting from "the hydrolysis of long chains of polypeptide residues in white connective tissue. Evidence '"hasiindi- -cated' that the primary'hydrolysis of collagentakes place at reactiveintervals-along these chains to produce an ideal parent undegraded gelatin molecule. This is'tollowed in varying -"degree by =a -=secondary hydrolysis at random intervals at the "less reactive bonds of the ideal parent gelatin molecule. Thisrexplains" howthe degradationreice .2 action is responsible for the random heterogeneous molecular pattern of any particular sample of gelatm.

Thus-the asymmetry of the gelatin molecule, together "with the heterogeneous molecular pattern, imparts the inherent properties of gel formation and viscosity to solutions-of gelatin prepared for'intravenous administration.

It 'has'been shown that the various molecular sizes donot function uniformly' in the circulation, neither are they retained with an equal degree of eifecti-veness.

It is generally accepted that gelatin proteins are true amphoteric electrolytes with ionization and physicochemical properties depending upon the pH value of their solutions. It is also a fact that every protein including gelatin has a definite isoelectric point at which ionization and there-fore physical and chemical reactivity are minimal. Some .of :these properties which show a minimum' influence at the isoelectric point include solubility, viscosity, colloid osmotic pressure and chemical activity. Thus, the'collo'id osmotic pressure of a gelatin solution will beat a minimum at the isoelectric point.

' :Since the dsoe'iectric point of-gelatin is influenced by the relative proportions of the free amino and free carboxyl groupings which provide the reactive ionic groupings of the proteins, any alterations in thisproportion will change the physicochem'ical properties materially.

lt is-an object of :our invention to provide stable chemical addition pro'ducts, or 'adducts, with gelatin that are characterized by --'irnproved -properties for various purposes including 'use as bioodplasma'substitutes or blood extenders.

It is a further object of our invention to provide a process of preparing adducts with gelatin involving alteration ofthe-ra'tio of free amino tofree carboxylic groups so as to decrease the isoelectric points of the resulting adducts as compared to gelatin and thereby improve the colloid osmotic function of such gelatin .materials for use as blood plasrna'substitutes or blood extenders.

It is a stiil further object-of our invention to provide a process of fractionating and recovering in a relatively pure state selected fractions of the chemical addition products or adducts, resulting from the reaction of gelatin with certain chemical compoundsas moreparticularly hereinafter described.

We have found that anhydrides of'tcertain.polycarboxylic acids, especially the following: succinic anhydride, citracoriic anhydride, itaconic anhydride, aconitic anhydride, and 'maleic anhydride, and also the chlorides of certaindicarboxylic acids, especially the following: succinyl chloride (butanedioyl chloride.) and 'fumarylfchloride react to form stable addition products or derivatives of gelatin which can beseparated and purified from undesirable products of the reaction by making appropriate use of their distinct-isoelectric properties as hereinafter described.

We have further found'that it'is possiblebyappropriate selection of the gelatin starting material so as to insure "that it will be composed of gelatin molecules falling within a-predetermined relatively narrow and appropriately selected averagemolecular sizerange, to. produce gelatin addition products byreaction with polycarboxylic acid-anhydrides and .dicarboxylic acid chlorides that may molecular sizerange of:aboutjl5,000 to ,36,090 is. reacted with "one ofthe aliphatic polycarboxyliqacid anhydrides or "dicarboxylicacid chlorides. above mentiouedflagelatin addition-product is produced which'mayabe separated by appropriate fractionat-ing and purifying "procedure from pared by reactir g the various carboxylic acid compounds other'products of the reaction and recovered in the form n though it may also be separated by filtration at the low of a pyrogenand antigen-free non-toxic material suitable temperature at which it is deposited. This precipitated for use as a blood plasma extender. material may be further refined by redissolving in water 'rWe have ,also 'found that the elsewhere described deheated to room temperature, then adjusting to a pH rivatives of gelatin resulting from1 reactions with dicar- 5 of about 3 and thereafter reprecipitating by the addition boxylic acid anhydrides such; as succinic anhydride are of suitable amounts of sodium chloride.

variable in composition and toxic within any single prep- The filtrate from this fraction contains still smaller aration asrbased upon chemical analysis, physical chemimolecular weight material which may be recovered by cal behavior and physiological reactions. They contain t e addition of Suitable amounts of sodium chloride. physiologically toxic materials which cannot be removed The resiliiing Precipitated maiei'id1 fip a iilil'ci without the isoelectric precipitation and fractionation fraction y iefind y I'di$$iViI1g inwaiel', j procedure di lo d herei 7 h V d 3n 7' V 7 lug the pH to bring it within the range 1.8-2.8 and then: The gelatin d i i f out i ti may b adding a suitable amount 7 of sodium chloride. The

mother liquors after separation of this third fraction conde c ib d herei b f ith gelatin of a d t i d tain recoverable products which have been precipitated m l la {Sizer rang; Ih :[eaction e d satigfagas iron compounds reactive with ammonium sulphide as torily at a pH of 6l0 and over a, temperature range of Wail as i disodiilm p p f C-.to 40 .0; 1r higherternperatpres are used the re- Y The p y addition products obtainedrby reading action will proceed more rapidly, but care *shouldbe a specific molecular weight fractionof gelatin with a t k tQ m h a i i clisconfinugd as soonras h 20 polycarboxylic acid anhydride or a dicarboxylic acid rea ti i Complete {pl gvioidfror i i i d d chloride suchas succinic anhydride or succinyl chloride tion of the gelatinwhich would lead to further reduction are Compounds having higher moiecilial Weights than in the number average 1 i h of m l i the gelatin used. Themoleculesare also more symmetri- V conlpcnent 511 reaction i Appropriate d i- V cal and have an increased number of ionizable carboxyl tions of sodium hydroxide solution or its equivalent are gifoi-ipsewhicil S fi y iowersthe isoeieciric Poilli of added i iti lly d dul'ing thg ecurse f he reaction the gelatin derivative or addition product below that of V maintai th d i d H di i j the gelatin itself. From the standpoint of their use as When th a' ti h gone to l i the reblood volume extenders-the more symmetrical type of atio Rmdueig maybe segara'tedby acidifyingfthe molecule represented by the gelatin derivatives above tune t the point f maximum i l biii hi h i mentioned has been. shown to bemuch better retained erally aboutLo t 15 H unit's b l h true i l 'in the intact bloodv vascular system of either thenormal tric i t f th d i d l i derjvan'irg Th i l for experimental shock animal. than is an unmodified which separates from the reaction mixture at the point gelatin; A150, file ilumbaf 0f ionizable groups ill h of maximum i i bili hi h generally i i hi a PH addition productLhave been so increased that at pH 7.3

range f t 5 varying with the particular poly. theeifective colloid osmotic pressure is significantly incarboxylic acid compound used to react with the gelatin, r creElsed is animmiscible, high density syrup which separates as V The principal features of these gelatin derivatives which sharply d fi d oil like layer in the bottom f the j makeflthem valuable as blood plasma extenders are as action. vessel. This material which represents the more follows: i i e i i I fully reacted substitution product as shown by its precise 40 .1 The highly asymmfiiric gelatin moicllie has been isoelectric properties is carefully separated from the made more symmeificai thus improving its p l y mother liquors which contain the lower molecular weight ieniioiia I i 'and incompletely reacted fractions of gelatin, also the The Productsdf a'higher number averagemolewler acid by-products of the reaction, by decantation, centrifu- {range P p y feactillgegfiiaiin Of an pp p 'gation or" other separatory methods. The immiscible se d l lsi e range with an aliphatic p 'syrupfor heavy oil fraction is further refined and freed h y i acid i y f f ii aiiphaiic diCaFbOXYi-iC acid t from lower molecular weight substitution products by f yappropriate fraciionation p l ng in distilled waterwith adjustment to a pHT Catlon as herein described; are non-toxic andfunction-as of 4 .to 6, followed by precipitation brought about by tefiective-cbiioid OSmOiiQVTagBH'ES-V' acidifying'to' attain the range of 2.0 to 3.5 with HCl; and -(3)= ased molecular symmetry of the heavy V addition of N ci, Thi i it ti Step maylbere- 'isoelejctricvfractionlmade from-predetermined molecular peated if necessary." This purification procedure is im- Size rangesiofigeiai-ifi' greatly decreases the iendemy i0 portant for the elimination of 'lowermolecular Weight 1' ,forinigels at refrigerated temperatures and at a P 'Y substitution products; incompletely reacted products and approximating ig g fli'desilabie degree acid lay-products whichprodu'ce abnormal :pharmacologiof fluidity and Overcoming 8 "of th obleFiions i0 cal and/or toxicefiects 'on intravenous administration. 'modifiedgelatinfas-a P1351113 Substitute P a y Alternatiye methodsof urifyi th heavy dditi 'imass Casualtieswhere:treatrnent for shock' must 'freproduct include reprecipitation-at approximately" pH? q yi fl fi the fi ld- 3 2-3.5 from methylyalc'ohol, ethyl alcohol, n-propyl alco- The fractionated il'eaciioilvprodilcis Ofigeiaiili a 11 01, isopropyl a1coho1 or a' 't e, The di 51 1 golthe compounds. described'herein are characterized by the 'rlde precipitated materialoccludes a certain amount of increase iiiililifibi iofeionizabie carboiililgfoillisi concentrated NaCl solution which .will provide a source "results, ill a 'Sigilificafli lowering Ofihe isoiectrii: Point of'thesodium chloride required f m ki fi i h d e which tmarkedly increases" the effective colloid osmotic saline solution (0.9% ,sodium l'chloridel of the gelatin i pres sureof the. gelatin: addition product at the pH value addition product when it is destined to be used as a blood J of extent-i0 which tiiE'iSOeiWiTiQPQiHtiS 'plasma'substitute or extender. A Any excess of occluded fired Farms-Somewhat w the ParticularPolycarboxyiic m y be removed by dialysis or other appropriate acidanhydride orfchloridereaction product. In the case procedure. H i r Qof theiumaryl chloride reactionproduct the isoelectric Theby-products remaining after the'primary precipitate poin t corresponds approximatelyto pH 3.8;- I tion and fractionation steps ofour process may be sub- The pllrifi at dn pro ethatwehave: found to be 7 ectedtoadditionalfractionating-treatments; They' have hseful. for obtaining new and eflectiveplasma volume been further separatedinto furtherfra'ctions. as follows; extenders or. plasmagsubstitutes'from the products ofthe f e lother liquors from th'e isoelectric separation T reaction of gelatin with the aliphatic polycarboxylic-acid are chilled to approximately 5 C, a flocculent precipitate compounds disclosed. herein, involves 1in 'generalthe 'pre i ii ii which iS fb e St jsolat edeby centrifugation; al- -,cipitation from the reaction :mixture of that material which appears :as an insoluble and immiscible syrup or oil at a particular pH within the range 2.0 to 3.5, depending on the starting material, redissolving the material in distilled water by adjusting to a pH of 5 to 7 with the use of NaOH and reprecipitation .at about pH 3 by the use of HCl and addition of appropriate amounts of NaCl. The product is dissolved and reprecipitated again if necessary to remove last traces of reaction products and/or smaller molecular weight derivatives which have markedly ditferent solubility properties. Following reprecipitation near the isoelectric point with addition of NaCl further purification may be effected by dialysis against Water through semi-permeable membranes, for example, cellulose acetate films. Another method of purification that .may be employed consistsin precipitation of the isoelectric gelatin addition product or derivative from its water solutions by use of methyl alcohol, ethyl alcohol, isopropyl alcohol or acetone.

The gelatin derivatives prepared, fractionated, and refined as above described to render them free from any 1 untoward reactions including pyrogenicity, antigenicity, or toxicity when put to parenteral use, are finally made into a solution of proper concentration, adjusted to the approximate pH of the blood and containing an isotonic concentration of sodium chloride or other such solution 1 of balanced physiological electrolytes. Further, the purified gelatin derivatives properly adjusted, may be suitably dried for reconstitution at a later time and in such manner as is physiologicallyindicated.

Although it is preferred to start with gelatin that has 1-- oeen partially degraded or otherwise brought to a number average molecular size within the range of about 15,000 to 36,000, it is to be understood that the fractionation and purification procedures described herein may be applied to reaction mixtures resulting from the reaction of a polycarboxylic acid compound of one of the kinds previously mentioned with undegraded gelatin or gelatins having a wider molecular size range or which are constituted of gelatin molecules falling within a difierent range than that above specified. When undegraded gelatin or a gelatin having a wider molecular size range is used .a more 'heterogenous mixture of reaction products will result, and it usually will be necessary to resort to a greater number of purification steps to obtain a satisfactory product. If a .gelatin starting material composed of gelatin molecules falling within a substantially different range is used, ap propriate adjustments of the degree of acidification may be required to bring the acidity of the reaction mixture to the .isoelectric point of the particular gelatin derivative sought to .be recovered.

The lowering .of the isoelectric point and reduction of the gelation temperature of gelatin has been emphasized herein relative to use of the addition products as a blood plasma extender. No distinction has been made between man and other'mammals since the extenders are intended for either. The addition product of gelatin with succinic anhydride has been extensively used and tested with human patients'with excellent results in raising and maintaining blood-pressure levels under shock conditions caused 'by surgery, accident, hemorrhage, and other causes.

The addition products of gelatin with succinic anhydride, 'itaconic anhydride, maleic anhydride and succinyl chloride have been tested and shown effective to raise the blood pressure of dogs after extensive induced bleeding and alsoefiective to cause the dogs to be able to withstand a second bleeding shortly after the first .if the first lost blood volume was replaced by an isotonic solution of one of the addition products. Since dogs are quite commonly bled in various laboratory experiments, and represent an expensive item in many researchprojects, a product that will quickly compensate for the lost blood is very useful f r th P rpose alone.

Moreover, there .are many other uses that may be made at the novel products of this invention that capitalize on their lower risoelectric points and decreased gelation prop- \erties.

Animal glue is a degraded collagen, i. e., gelatin, although generally not as highly refined as edible or medicinal gelatins. Generally urea or thiourea is added to the glue to restrain gelation before its ultimate use. It is thuspossible to-eliminate or decrease the amount of urea in the glue by using the products of this invention.

Gelatin is also used for sizing rayon but 'the gelatin solutions are of necessity very dilute to avoid gelation although it is otherwise desirable to use more concentrated solutions. More concentrated solutions are of course pos- 'sible without gelation if the present products are used.

It has-been found that formaldehyde does not afieot the products of this invention, probably because the free amino groups are combined with carboxylic groups of the anhydrides or chlorides. Therefore these new .products can be used in film coatings to get difierenti-al gelatins required for color films.

It has further been 'found that the products of the present invention are considerably more plastic than previously known gelatin. Therefore, these new products may be incorporated into various chewing gum bases such as polyvinyl acetate or chicle whereas regular gelatin has been considered too brittle for such use.

Along these same lines, these new products may be used as plasticizing agents in admixture with regular gelatin for the manufacture of soft gelatin capsules, thus eliminating the use of completely foreign substances such as glycerol.

The following examples illustrate how the new class of refined gelatin derivatives having improved colloid osmotic function for use as plasma substitutes are made:

Example .1

3.000cc. of a-6% solution of gelatin grams) having a .number average molecular weight of approximately 36,000 .(H =.36 at 55 C.) was held at a temperature of 36 C. and adjusted to pH 9 with a concentrated NaOI-I solution. 60 grams of powdered succinic acid anhydride was gradually added while stirring the gelatin solution. As the reaction proceeded the pH was maintained in the range of -8.5l0 by the regular addition of NaOH in a concentrated aqueous solution. After the addition of the succinic acid anhydride was completed and no further change in pH was evident, the reaction was regarded finished. The mixture was then acidified with 132 cc. concentrated HCl to pH 2.7. After a short time of settling an immiscible high density syrup like fraction separated as a clear layer in the bottom of the vessel. The supernatant liquid containing low molecular weight materials and reaction by-products was grossly separated by syphoning and the .heavy layer was more completely separated in .a sepratory funnel. The heavy oily fraction representing the desired adduct product was then dissolved in 2 liters of distilled water at pH 45-5 and then adjusted to pH 2.5-3.0 and precipitated by the gradual addition of 584 grams of solid sodium chloride to give an approximately 30% concentration of NaCl. The precipitate first dispersesthroughout the solution, then as the concentration of NaCl increases in the solution, the adduct forms a coagulum which floats. Care must be taken during final addition of salt to avoid enmeshing it in the coagulum. The solution containing sodium chloride and reaction products of smaller molecular weight was separated from the coagulum which was worked to allow encapsulated brine droplets to run voff as completely as possible. Substantially complete removal of the brine is necessary in orderto reach .a NaCl content at/or below the ratio of 1 part NaCl to 3.33 parts of purified addition product for allowing isotonic NaCl concentration .of 0.9% in the final intravenous preparation based on a 3% gelatin succinate addition product. The purified product was then dissolved sterile, .pyrogen free distilled water aided by use of iNaCl content. alysis for. removal of the reaction by products from the heavy fraction of the anhydride addition product precohol or acetone maybe used.-

gelatin starting 'rn aterial used.

concentrated NaO'I-I to yield a solutionat 7.0. This solution of the adductflwasfurther adjusted after analysis :to final 3% concentration in isotoni'clNaCl at pH 7.3. The yield of purified gelatin-succinate addition product was 105 grams or a 58.3% yield based on the starting quantity of gelatin used.- -The, solution was filtered, botcipitation by methyl alcohol, ethyl alcohol,'isopropylal- Example I The above procedure was repeated except that the reaction between the gelatin and succinic anhydride was carried out in the range of pH'5.5 6.5. The reaction mixture was then subjected to fractionation to obtain the desired addition product and this wassubjected to repeated purifying treatments in a similar manner as decarrying on the reaction'as described in Example 1.

Example 3 The procedure in Example 1 above was repeated except that gelatin starting material having a number average molecular-weight of approximately 20,000 (H .18

at 55 C.) was used. The heavy fraction of addition prodnct'gavea yield of 49.5% based on the quantity of The purified addition product gave a 3% solution in isotonic NaCl at pH 7.3 which was fluid at temperatures below 5 C.

Example 4 1500 cc. of a 6% solution of gelatin (90 grams) having a number average molecular weight of approximately 20,000 to 25,000 was adjusted to a temperature of 13 C. and to pH 9 with NaOH. 30 grams of itaconic anhydride was added slowly with stirring while maintaining the pH in the range of 9 to 10 .by addition of NaOH.

After the addition of the ita conic' anhydride was completed and no further change in the pH was evident, the reaction was regarded'finished; Concentrated HCl was added to the mixture to pH 3.5 where the separation of a heavy fraction was sharp and distinct.

solved with the aid of NaOH in one liter of distilled water at pH 3.95, and then reprecipitated at pH 3.5 by

adjustment of the pH with HCl. This reprecipitated gelatin-itaconic addition product gave a yield of 102% based on the starting gelatin weight. .It was thenbrought 'to a 6% concentration in isotonic NaCl atpl-I 7.3 and autoclaved at lbs. steampressure for minutes. It was then ready for intravenous administration. The 6% V The heavy fraction separated by use of a separatory funnel was dis scribed in Example 1 .The yield bf the desired gelatin I succinate was considerably less than that obtained in stitution product;

-solved'in sterile, pyrogen free distilled water aided byuse solution did not form a gel at 5 C. during '18 hours observation. v heavy fraction which could be precipitated at its isoelectric point from water solutions without NaCl addition. 1

Example 1500 'cc'. of a 6%"solution of gelatin grams) having a number average molecular weight-of approximately 15,000-'20,0 00 '(H =.14-..18 at 55 C.) was held at room temperature and adjusted to pH 9 with NaOH.

The condensation reaction gave a type of tures below 5 C;

' 30 grams of 'citraconic anhydridewas added slowly with stirring while maintaining the pH in the range of 9-10. The reaction mixture was acidifiedto pH'2.78' at which pointtheheavy fraction' separated sharply. -It was transferred to one liter -of distilled water-and'dis'solved therein by suitable adjustment of the pH value with the use of fconcentrated NaOH. After again acidifying to a pH of 2.78, this addition'product'was precipitated by addition of grarns-of NaCl. This gelatin citraconic anhydride addition product in 6% concentrationiin isotonic saline at pH 7.3 gave a gel at 5 C. after'18'hours. After, a second purification involving re-solution, readjustment of the pH to 2.78 and precipitationby addition of salt, the product in concentrated aqueous solution at pH 7 was dialyzed in Visldng tubing against running'water for two hours to reduce thesalt'content below 0.9 grarnper 6 grams of gelatin addition product. i

7 Example '6 V p 3000 cc. of 6% solution of gelatin grams) having a number average molecular weight of approximately 20,000-(H =0.18) were held at a temperature of 28- 37 C. and adjusted to pH 9 with concentratedNaOH solution. 60 gramsof succinyl chloride were gradually .added while stirringth'e' gelatin solution. As the reaction proceeded the pH was maintained in the range of 85-10 by the regular addition of concentrated NaOH solution until 128 cc. had been added. After the addition of the succinyl chloride was completed and no further change in pH was evident, the reaction was'regarded finished. The. mixture wasthen acidified with 89 cc. concentrated HCl to pH'2.3. After a short timefofsettling, an immiscible,

high density, syrup like fraction separated as a clearlayer in the bottom of the vessel. The supernatant liquid containing low molecular weight materials and reaction byproducts wasfgrossly' separated bysyphoning and the heavy layer further .or more completely separated in a separatory funnel. The'heavyoily fraction representing the desired primary gelatin derivative was dissolved in 1.5 liters of distilled water. and separated as a 'coagulum by thefgradual additionof 607.. grams: of solid sodium chloride at pH 3.0. The precipitate: first disperses throughout the solution, then as the. concentrationof NaCl increasesf in'the solution, the gelatin derivative forms a coaglum; Care must be taken during final addition of salt to avoid enme'shing it in the cagulum; The solution of'sodium chloride-containing reaction products of smaller molecular weight was decanted leaving the coaglum which was worked to allow encapsulated brine dropletsto r un off as completely as possible. Rather complete removal of the brine ivasnecessary; in orderto reach a salt content at/orbelow the ratio ofl part NaCl to 3.33 parts of purified product for allowing isotonic NaCl concentration'of 0;9%- in the final intravenous preparation based on a 3% gelatin succinylchloride sub- The purified product was .then disof 'concentratedNaOH solution to yield-a solution at pH 7.0. This solution'was further adjusted,.after analysis to 'a'final 3% gelatin derivative solution in 0.9%1NaCl at pH 7.3. The yield ofrpurified gelatin succinyl chloride product was 94 grams or a 52.2% yield based on the starting quantity of gelatin used. The solution was filtered, bottled in 500v cc..bo ttl es and sterilized at,15 lbs.

steam pressure for 20 minutes when it was ready for'intravenous administration; The productin 3% concentration ina' 0.9% NaClsolution remained fluid at tempera- 3000 a. of a 6% solution of gelatin (rl80 grams) haw ing a number .average molecular weight of'approximately 20,000 (H5=0.18) were held at a temperature of 22- 30C. and adjusted 'to pH 9 with NaOH's'olutiori.

anhydride and said mixture is acidified to a pH of approximately 28.

13. A gelatin derivative consisting of that'fraction of the products resulting from reacting gelatin having a number average molecular Weight within the range 15,000 to 36,000 with a polycarboxylic acid compound selected from the group consisting of succinic, citraconic, itaconic, aconitic and maleic anhydride and succinyl and fumaryl chloride in an aqueous medium adjusted to a pH of 6.

' to and precipitating as the coagulum from the reaction said compound is maleic anhydride and said mixture is acidified to a pH of approximately 3.

' 17. A gelatin derivative as claimed in claim 13 wherein said compound is succinyl chloride and said mixture is acidified to a' pH of approximately 2.3.. a

18. A gelatin derivative as claimed in claim 13 wherein said compound is citraconic anhydride and said mixture is acidified to a pH of approximately 2.8.

19. A gelatin derivative consisting of that fraction of the products resulting from'reacting gelatin with a polycarboxylic acid compound selected from the group consisting of succ'inic, citraconic, itaconic, aconitic and maleic anhydride and succinyl and fumaryl chloride in an aqueous medium adjusted to a pH of 6 to 10, and precipitating as a coagulum from the reaction mixture upon acidifying said mixture to a pH range from about the isoelectric point of said fraction to 2 pH units'therebelow.

20. A composition of matter suitable for use as blood plasma extender consisting of a dispersion in physiological saline of a gelatin derivative resulting from the'reaction of gelatin having a number average molecular range within the range 15,000 to 36,000 with a polycarboxylic acid compound selected from the group consisting of succinic, citraconic, itaconic, aconitic and maleic anhydride and s'uccinyl'and fumaryl chloride inan aqueous medium adjusted to a pH of 6 to l0 and which is precipitated as the coaglum from the reaction mixture upon acidifying said mixture to 'a pH range from about the isoelcctric point of said derivative to 2 pH units therebelow, said gelatin derivative before it is incorporated'with the physiological saline having been purified by suitable working, Washing and reprecipitation of said coaglum to remove therefrom any incompletely reacted gelatin and any uncoagulated reaction products, said composition being free from physiologically toxic materials and remaining-fluid at temperatures below 5 C. when existing as 3-6% concentrations of said gelatin derivative in isotonic NaCl at pH 7.3.

21. A composition as claimed in claim 20 whereinsaid compound is succinic anhydride and the acidity of said mixture is adjusted to approximately.pH2.7."

' 22. A'composition as claimed in claim'20 wherein said compound is itaconic anhydride and the acidityrof said -mixture is adjusted to approximately pH 3.5.

. 23. A composition as claimed in claim 20 wherein ;said compound is maleic anhydride and the acidity of said mixture is adjusted to approximately pH 3.

24. A composition as claimed in claim 20 wherein said compound is succinyl chloride and the acidity of said mixture is adjusted to approximately pH 2.3. V 25. A compositionas claimed in claim 20 wherein said compound is citraconic anhydride and the acidity of said mixture is adjusted to approximately pH 2.8.

References Cited in the file of thispatent UNITED STATES PATENTS 2,178,569 Ferguson Nov. 7, 1939 2,518,666 Damschroeder et al. Aug. 15, 1950 7 2,525,753 Yutzy et al. ....a Oct. 10, 1950 FOREIGN PATENTS 831,630 France Sept; 9, 1938 e 7 OTHER REFERENCES p Loewe et al.: Am. J. Med. Sci., pp. 54-63, p. 55 pert.,

July 1944.

Amberson: Biol. Reviews, pp. 48, 79, pp. 76 and 77 pert., January 1937. 

13. A GELATIN DERIVATIVE CONSISTING OF THAT FRACTION OF THE PRODUCTS RESULTING FROM REACTING GELATIN HAVING A NUMBER AVERAGE MOLECULAR WEIGHT WITHIN THE RANGE 15,000 TO 36,000 WITH A POLYCARBOXYLIC ACID COMPOUND SELECTED FROM THE GROUP CONSISTING OF SUCCINIC, CITRACONIC, ITACONIC, ACONITIC AND MALEIC ANHYDRIDE AND SUCCINYL AND FUMARYL CHLORIDE IN AN AQUEOUS MEDIUM ADJUSTED TO A PH OF 6 TO 10 AND PRECIPITATING AS THE COAGULUM FROM THE REACTION MIXTURE UPON ACIDIFYING SAID MIXTURE OF A PH RANGE FROM ABOUT THE ISOELECTRIC POINT OF SAID FRACTION TO 2 PH UNITS THEREBELOW. 